Genetic information encoded in DNA molecules is expressed by a series of steps involving transcription of DNA into mRNA and the subsequent translation of the mRNA into polypeptides or proteins. The expression of the encoded information to form polypeptides is initiated at the promoter site, a region on the DNA molecule to which RNA polymerase binds and initiates transcription.
Recombinant production of proteins and peptides has become a hallmark of the biomedical and industrial biochemical industry. One of the factors influencing the cost of commercial protein/peptide production is the efficient expression of the desired gene product. Factors influencing the efficiency of the process include, but are lot limited to gene dosage (i.e. copy number), promoter strength, and the ability to control expression (i.e. inducibility).
Historically, one means to increase protein production has been the use of multi-copy plasmids. However, the increased metabolic burden placed on the cell often results in a decreased growth rate and plasmid instability. As such, it is desirable to use a strong promoter so that the copy number is minimized. The use of a strong promoter facilitates increased protein production while minimizing the metabolic burden on the host cell (i.e. fewer copies of the gene targeted for expression are required to achieve the same level of protein yield).
The use of strong promoters often requires a level of control when expressing the desired gene product. Uncontrolled constitutive expression often results in undesirable effects on the growth and/or viability of the recombinant host cell. As such, the use of strong, inducible promoters is desired. Preferably, the promoter used is characterized by tightly regulated expression and is induced using a condition or compound that is safe, environmentally friendly, and economical.
The araB gene and its promoter (“araB promoter” also known as the PBAD promoter) are located in the L-arabinose operon. The endogenous L-arabinose operon has been studied in various microorganisms including, but not limited to Escherichia coli, Salmonella typhimurium, and Bacillus subtilis ((Horwitiz et al., Gene (1981) 14:309-319; Lin et al., Gene (1985) 34:111-122; Lin et al. Gene (1985) 34:123-128; Lin et al., Gene (1985) 34: 129-134); Schleif, R., Trends in Genet. (2000) 16(12):559-565; U.S. Pat. Nos. 5,028,530; and 6,030,807). The operon is comprised of 3 structural genes (araA, araB, and araD) encoding enzymes responsible for converting L-arabinose to D-xylose-5-phosphate. The gene araA encodes the enzyme arabinose isomerase, responsible for converting arabinose to ribulose. Ribulokinase (encoded by the gene araB) phosphorylates ribulose to make ribulose-5-phosphate. The enzyme ribulose-5-phosphate epimerase (encoded by the gene araD) converts ribulose-5-phosphate to xylulose-5-phosphate, which can be metabolized via the pentose phosphate pathway. The araBAD operon is coordinately controlled by the inducer L-arabinose and the AraC regulatory gene product (Guzman et al., (1995) J. Bacteriol. 177:4121-4130). PBAD based expression systems based are widely used and commercially available from companies such as Invitrogen (Carlsbad, Calif.).
The PBAD expression system is tightly controlled and the inducer, L-arabinose, is safe and economical. However, the wild type araB promoter is not generally considered a strong promoter once induced. As such, use of the currently available PBAD-based expression systems is often unattractive for low cost peptide/protein production where optimal protein yield is desired.
The problem to be solved is to provide an arabinose inducible expression system having the ability to increase protein yield when operably linked to a coding sequence of interest.